DNA is damaged by many chemical and physical environmental agents. Most damage is removed by one of the cellular DNA repair systems, but some persistent damage blocks DNA replication or causes errors in DNA replication, leading to mutations and altered function. Is the persistence of this damage the result of random oversight by the repair systems or does it reflection fundamental properties of the damaged site? In the bacterium Escherichia coli, most large bulky adducts are repaired by the uvr system, including adducts formed by N-acetoxy-N-acetyl-2-aminofluorene (-AAF) and N-hydroxy-N-2-aminofluorene (-AF). However, indirect evidence suggests that -AAF adducts within certain mutation-prone sequences are poorly repaired. The presence of the adduct in these sequences may induce a localized transition to Z-form DNA. In this project, the interaction of the UvrABC proteins with -AAF adducts and -AF adducts in mutation-prone and normal sequences will be examined directly in vitro. High resolution gel electrophoresis will be combined with specific chemical and enzymatic reactions to determine quantitatively the sequence distribution of DNA damage, UvrA binding and UvrABC incision. This method will allow sequence-specific adduct structures that interact unusually with the repair system to be identified and will allow an exploration of the underlying structural features. The sensitivity of DNA conformational transitions to variation in the nature and concentration of salts in the solution will be exploited to probe the physical basis for altered interactions with the repair proteins. Although the Uvr system uses distortion in the DNA helix to detect damage, only a specific form of distortion may be recognized. Damage inducing unusual distortions in the DNA may escape detection by the repair system. Since the Uvr system of Escherichia coli has elements in common with repair systems in higher organisms, the results of this study may illuminate specific connections between the persistence of damage in DNA, DNA repair and mutagenesis or carcinogenesis.